RNA-directed DNA Methylation and Heterochromatinization at the MPF.

(A) Genomic structure of the FLC locus and flanking regions examined by bisulfite sequencing (B1, B2, B3 and B4) or ChIP (C1, C2 and C3). Green box represents the hAT element; pink boxes represent exons; the gray arrow represents the promoter; the orange box represents the TE insertion in Ler. (B) Small RNA tags matched to MPF found from the MPSS (green) or 454 sequencing data (red), and the LNA probe used for small RNA hybridization (blue) are represented with their length indicated by numbers. The color coding of the cytosines in (B) matches the legend in (C). (C) Bisulfite sequencing result of the MPF at the B1 region in Ler. The bars with red stars represent sites that were detected by Southern blot (Figure 1) and n indicates the number of the sequenced clones. (D) Small RNA Northern blots probed with the LNA probe (B) in Ler and Col; tRNA and other RNA bands stained with ethidium bromide (EtBr) were used to indicate the amount of loaded RNA. (E) Chromatin Immunoprecipitation (ChIP) to detect H3K9 mono-, di-, and tri-methylation (represented as H3K9me1, H3K9me2, and H3K9me3, respectively) at MPF (C1) in Ler and Col. Input is saved before immunoprecipitation and “No AB” refers to the sample without antibody. Ta3 served as an internal control for heterochromatic loci.